分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.029

生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 107-114.doi: 10.13560/j.cnki.biotech.bull.1985.2016.11.029

分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物

姜文灿^{1, 3}, 岳素文², 江洪², 葛素君¹, 王成彬^{1, 3}

1. 中国人民解放军总医院临检科,北京 100853;
2. 北京泰格瑞分子检验有限公司,北京 100085;
3. 温州医科大学检验医学院生命科学学院,温州 325000

收稿日期:2016-03-02 出版日期:2016-11-25 发布日期:2016-11-11
作者简介:姜文灿,男,硕士研究生,研究方向：分子生物学;E-mail：jiangwencan2014@126.com
基金资助:
国家重大科学仪器设备开发专项（2012YQ03026107）

New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR

JIANG Wen-can^{1, 3}, YUE Su-wen², JIANG Hong², GE Su-jun¹, WANG Cheng-bin^{1, 3}

1. Department of Clinical Laboratory Medicine,Chinese People's Liberation Army General Hospital & Postgraduate Medical School,Beijing 100853;
2. Beijing Tag Array Molecular Test Co.,Ltd,Beijing 100085;
3. College of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325000

Received:2016-03-02 Published:2016-11-25 Online:2016-11-11

摘要/Abstract

摘要： 在TaqMan探针法实时荧光定量PCR中,排除由引物探针聚合延伸引起的假阳性问题以及由引物二聚体（Primer Dimer,PD）引起的假阴性问题。首先对不同探针进行引物探针聚合实验;然后通过双重PCR的干扰实验选择合适的内标基因,并使用内标质粒检测验证中部同序引物排除PD干扰的实用性;最后比较探针法不同体系检测HBV基因的灵敏度。结果表明,引物与探针聚合实验中,含有反义碱基的HBVP4在重复实验中未出现假阳性;竞争性双重PCR和非竞争性双重PCR两模板开始出现干扰作用的浓度差分别为20倍和100倍;对于内标基因,使用中部同序引物对以及普通引物对分别可以检测到10^-9和10^-8稀释度;3种HBV基因检测体系,使用普通引物对可以检测到Ct33左右,加入内标系统和使用中部同序引物对均可检测到Ct35左右。在TaqMan-分子信标结构调整的基础上引入反义碱基可以在排除由引物和探针聚合延伸引起的假阳性问题;在探针法中,使用中部同序引物对和加入内标系统均可降低PD的干扰程度,提升检测的灵敏度。

关键词: 实时荧光定量PCR, TaqMan探针, 反义碱基, 引物二聚体, 内标系统

Abstract: Our purpose is to eliminate the false positive problem caused by primer-probe aggregation and extension,as well as the false negative problem caused by primer dimer（PD）. First,the primer and probe aggregation results were tested for different probes;then the appropriate internal control gene（IC）was selected based on the competitive interference experiments of duplex PCR,simultaneously,the practicability of central-home primer excluding the inference of PD was verified on the plasmid of the IC;and finally,the sensitivities of different systems for TaqMan method were compared. The probe HBVP4,containing an antisense base,did not produce false positive results in the repeated trials;the concentration difference at which interferences for the templates of competitive and non-competitive duplex PCR were noticed was 20 times and 100 times,respectively. The dilution that could detected by central-home primer and common primer was 10^-9 and 10^-8,respectively. And for 3 HBV gene detection systems,the detection could be until Ct33 if using common primer,and Ct35 if using central-home primer and by the accession of IC. Based on adjustments of TaqMan-Molecular Beacon and introducing antisense,the false positive problem caused by primer-probe aggregation and extension was excluded. In probe method,the use of central-home primer and IC could both reduced the influences caused by PD,and therefore increase the sensitivity of detection.

Key words: real time fluorescent quantitative PCR, TaqMan probe, antisense base, primer dimer, internal control

姜文灿, 岳素文, 江洪, 葛素君, 王成彬. 分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物[J]. 生物技术通报, 2016, 32(11): 107-114.

JIANG Wen-can, YUE Su-wen, JIANG Hong, GE Su-jun, WANG Cheng-bin. New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR[J]. Biotechnology Bulletin, 2016, 32(11): 107-114.

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分子信标-TaqMan探针法实时荧光定量PCR新型探针及引物

New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR

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